Methods for detecting phosphorylation of peptides and proteins is an area in the fields of analytical chemistry, medicinal chemistry, and biochemistry where considerable effort has been expended. Considerable efforts have also been made with respect to methods for detecting other post-translational modifications of peptides and proteins such as acylation, glycosylation, alkylation, and adenylation.
Post-translational modification of proteins has been recognized for decades as a significant mode of regulation. In particular, phosphorylation, and the reverse process, dephosphorylation, are key factors in numerous aspects of cell signaling, cell cycle regulation, and response to stress (reviewed in Yan et al., J. Chromatography A, 808:23-41 (1998)). Phosphorylation of proteins is catalyzed by a class of enzymes called protein kinases, which transfer the terminal phosphate from adenosine triphosphate (ATP) to a given amino acid residue, typically serine, threonine, or tyrosine. In general, phosphorylation is a reversible process. Dephosphorylation is carried out by protein phosphatases. Moreover, kinases and phosphatases may be inhibited by various factors (Hidaka et al., Biochemistry 23:5036-5041 (1984)). Because of their importance in cell signaling and cell cycle regulation, proteins in the phosphorylation cycle, both enzymes and their substrates, have become major targets for the development of pharmaceutical compounds. Protein kinases, in particular, because of their role in cell division and cancer progression, have emerged as the principal targets of drugs aimed at treating cancer, immunosuppression, retinopathy, rheumatoid arthritis, and neurodegeneration (Cohen, Nature Reviews Drug Discovery, 1:309-315 (2002)).
A variety of methods exists for monitoring and detecting the phosphorylation state of proteins, which is important, among other purposes, for assessing the efficacy of candidate pharmaceutical agents. Antibody-based detection is among the most widely used of these methods. In addition to monoclonal antibodies specific for individual proteins, more recent endeavors have resulted in the production of phospho-motif antibodies, which recognize a phosphoserine or phosphothreonine residue in a conserved amino acid motif (reviewed in Berwick et al., Trends in Biochemical Science, 29:227-232 (2004)). Generation of such antibodies requires extensive characterization of the substrate specificity of the kinases being examined. Alternative methods for monitoring kinase activity make use of 32P-radiolabeled phosphate groups and mass spectrometry to identify modification in protein composition before and after treatment with a kinase (Yan et al., Journal of Chromatography A, 808:23-41 (1998)).
In addition to phosphorylation, other co- and post-translational modifications are known to exert regulatory effects on proteins. Acylation, particularly by either fatty acyl or prenyl residues being covalently linked to an —SH group of a cysteine residue, is one such modification (Kendrew, J. editor, THE ENCYCLOPEDIA OF MOLECULAR BIOLOGY, Blackwell Science, Inc. Cambridge, Mass., 1994, p. 15). Ras proteins undergo several post-translational modifications, including farnesylation. Inhibition of the enzyme that carries out this modification, farnesyl transferase, is a promising approach to controlling this oncogenic protein (Crul et al., Anticancer Drugs. 12(3):163-84 (2001)). Other commonly encountered post-translational modifications, such as glycosylation and proteolytic cleavage, are important in protein secretion and translocation.
Although various methods have been used to detect phosphorylation and other co- and post-translational modifications of peptides and proteins, a need exists for simple devices and methods that may be used to rapidly detect such modifications, particularly the phosphorylation of peptides and proteins without the need for radioactive labeling and other manipulation, such as hybridization and washing, and without the need for complex instrumentation. A need also remains for methods of manufacturing devices for use in differentiating between post-translationally modified peptides and peptides. Also needed are rapid, high throughput methods for directly detecting phosphorylation state irrespective of the identity of the modified amino acid or its location within a particular protein sequence or known kinase motif.